Vinyl acetate, immobilization enzymes

A novel continuous-flow SCCO2 process for the kinetic resolution of 1-phenyethanol enantiomers (Figure 30) using Novozym 435 immobilized enzyme from Candida antarctica was described by Matsuda et al. [51], The lipase enzyme, selectively acetylated the R)-alcohol component. A mixture of starting material and vinyl acetate was passed through the enzyme with supercritical carbon-dioxide (Figure 31). The reaction zone was pressurized and heated, so the reaction could be performed imder supercritical conditions, synthesizing the desired (i )-acetate with 99.7% ee. and 47% yield. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Such ILCE showed remarkably high enantioselectivity for transesterification reaction of several secondary alcohols in the presence of vinyl acetate in toluene, and there was no significant deterioration in activity even after multiple (five times) use. Horseradish peroxidase (HRP) is another example to be coated with both hydrophobic and hydrophilic ILs such as [BMIMJITf N] and [BMIM][PEg] toward enzymatic synthesis of polyaniline [23]. An attractive point of the ILCE-based immobilization method is easy recovery of enzyme/IL phase by liquid-liquid phase separation and effective reusability of the enzyme (Eigs. 10.8, 10.9) [23, 59]. 

An interesting electrochemical method for the determination of bound sialic acid has been developed, making use of a potentiometric four-channel thick-film sensor [236]. The sialidase sensor consists of a bilayer of a membrane containing Clostridium perfringens sialidase immobilized in a poly(vinyl acetate)-polyethylene copolymer, which is placed on top of an fT -selective poly(vinyl chloride)-poly(vinyl acetate) indicator membrane. The enzyme-induced release of bound sialic acid leads to a concomitant decrease in pA a of the carboxyl function of sialic acid. This decrease affords a local pH change inside the sialidase-containing sensor membrane, which is monitored by the H -selective indicator membrane. The pH optimum of the sialidase sensor was pH 4 for sialyllactose, mucin and colominic acid. 

Enzymes other than CAL B have also been reported to operate under the biphasic conditions. CAL A and CAL B or a lipase from Mucor miehei were tested for the kinetic resolution of glycidol using vinyl acetate or vinyl butyrate. The enzymes were used either suspended in the free ILs or immobilized, when the reactions were carried out in [EMIM][NTf2] [71]. CALAwas inactive, butthe other two enzymes showed activity, albeit at 10-20% of that in the absence of COj whether they were free or immobilized. In general, the supported enzymes showed superior performance [71]. Chymotripsin, a specific protease for aromatic amino acids, was found to catalyze the hydrolysis or transesterification of the ethyl ester of N-acetyl-phenylalanine with propanol in scC02-[RMIM][PF5] (R = butyl or octyl) with or without added water [Eq. (15)]. 

The use of an active carbon cloth (Kynol AGG 507-15) with a large surface (1500 m g ) has also been reported for the immobilization of a lipase from Burkholderia cepacia (Amano PS, enzyme immobilized on Gelite) [27]. The system has been studied for the KR of several sec-alcohols with vinyl acetate as the acyl donor, using conventional solvents in a batch system (Figure 17.2a). A comparison of the catalyst coated and uncoated by an IL was made, showing that the activity and stability for reuse were preserved only for the system coated with [EMIM][NTf2]. Gonversions of 50% were obtained, with values of 98% ee for this system, with toluene as the solvent... 

An interesting example of lower rim selective functionalization is the regioselective acylation of the C(2) side chain of tetraalcohol 25b, obtained using immobilized lipase from Mucor miehei in organic solvents and vinyl acetate as acylating agent. The enzyme also proved capable of partially acetylating the prochiral side chains at C(8) and C(20), but did not produce the desired dissymmetrization [19]. 

A general procedure for enantioselective acylation of an alcohol with enol acetate or sucdnic anhydride is as follows. Dissolve 1 g of substrate alcohol in solvent (35 mL of hexane, toluene, or tetrahydrofuran) containing vinyl or iso-propenyl acetate (3 mL) and triethylamine (0.5 mL) (see following note). Add 0.5 g of hydrolase powder or immobilized hydrolase the enzyme powder will not dissolve Stir the suspension with a magnetic stirrer and monitor the progress of... 

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